Regulation of RANKL‐induced osteoclastogenesis by TGF‐β through molecular interaction between Smad3 and Traf6

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling. © 2011 American Society for Bone and Mineral Research.     

Orthopedic wear debris mediated inflammatory osteolysis is mediated in part by NALP3 inflammasome activation

Activation of myeloid cells by orthopedic particulate debris is a key event in the pathogenesis of periprosthetic osteolysis and implant loosening after total joint replacement (TJR). Several lines of evidence implicate NACHT, LRR, and PYD domains‐containing protein 3 (NALP3) inflammasome‐mediated production of interleukin 1 beta (IL‐1β) in the pathogenesis of clinical disorders ascribable to foreign particulate materials, including asbestos, silica, and urate crystals. Recent reports indicate that orthopedic polymer products and metallic particulates and ions may activate the same pathway. Here, we investigated the contribution of the NALP3 inflammasome to the pathogenesis of peri‐implant osteolysis. Pharmaceutical and genetic perturbations of caspase‐1 and inflammasome components were used to assess the role of the NALP3 inflammasome in IL‐1β production and osteoclast formation by human monocytes and mouse macrophages in response to polymethylmethacrylate (PMMA) particle phagocytosis. The role of caspase‐1 in a mouse calvarial model of particle‐mediated osteolysis was assessed using µCT. Phagocytosis of PMMA particles induces caspase‐1 dependent release of IL‐1β from human monocytes and mouse macrophages. Importantly, using macrophages from mice deficient in components of the NALP3 inflammasome, we show PMMA‐induced IL‐1β production is strictly dependent on these components. Mice lacking caspase‐1, the sole effector of the NALP3 inflammasome, show reduced orthopedic wear particle‐induced calvarial osteolysis compared to wild‐type controls. Absence of NALP3 inflammasome components fails to alter osteoclast formation in vitro. Our findings identify the NALP3 inflammasome as a critical mediator of orthopedic wear‐induced osteolysis and as a viable therapeutic target for the treatment of periprosthetic osteolysis. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:73–80, 2012     

Antisense oligonucleotide targeting TNF‐α can suppress co‐cr‐mo particle‐induced osteolysis

The most common cause of implant failure in joint replacement is aseptic loosening due to particle‐induced osteolysis. TNF‐α has been shown to be one of the key factors in the process of osteoclastogenesis. Anti‐TNF agents are useful in the treatment of joint inflammation related to osteolysis. This study investigated the effect of a single subcutaneous dose of an antisense oligonucleotide (ASO) on particle‐induced osteolysis. We utilized the murine calvaria osteolysis model in C57BL/J6 mice. Bone resorption was measured by the toluidine blue staining. Osteoclasts were detected by tartrate resistant acid phosphatase (TRAP) staining assay and were quantified by a TRAP quantification kit. Results show that bone resorption is 0.347 ± 0.09 mm² in mice with particle implantation, and decreased to 0.123 ± 0.05 mm² and 0.052 ± 0.02 mm² after ASO treatment with low and high doses, respectively. The number of osteoclasts in animal calvaria treated with ASO is reduced compared with that of untreated animals, and the quantification results indicate that about 90% of osteoclastogenesis is suppressed by the ASO. In addition, the osteoclastogenesis can be reestablished by the addition of TNF‐α. In conclusion, we demonstrate that the antisense oligonucleotide targeting to TNF‐α can suppress osteolysis induced by metal particles in a murine calvaria model. This new finding may be of value in the search for novel therapeutic methods for implant loosening. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1114–1120, 2008     

TNF‐α–induced NF‐κB signaling reverses age‐related declines in VEGF induction and angiogenic activity in intervertebral disc tissues

We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF‐α induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF‐α–induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF‐α–induced VEGF using organ disc cultures with wild type, TNF receptor 1‐null (TNF‐RI^null), or TNF receptor 2‐null (TNF‐RII^null) mice. VEGF induction was inhibited when we used TNF‐RI^null‐derived disc tissues. NF‐κB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF‐α induced VEGF expression in disc cells primarily through the NF‐κB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF‐α stimulation. TNF‐α treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF‐κB inhibitors or anti‐VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF‐κB pathway, both of which are induced by TNF‐α. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patient's age. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:229–235, 2009     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

LPS‐Induced Inhibition of Osteogenesis Is TNF‐α Dependent in a Murine Tooth Extraction Model

TNF‐α is a major etiologic factor of inflammatory bone diseases such as periodontitis and rheumatoid arthritis. In addition, patients with metabolic diseases such as chronic heart disease and diabetes have significantly increased plasma levels of TNF‐α. Several lines of evidence show inhibition of osteoblastogenesis by TNF‐α in vitro. Therefore, bone formation and osteogenesis in these patients might be inhibited because of TNF‐α. However, little is known about the inhibitory role of TNF‐α in bone formation/osteogenesis in vivo. The purpose of this study was to investigate the role of TNF‐α in osteogenesis using a murine tooth extraction model. Lipopolysaccharide (LPS) was injected subcutaneously into the calvariae of either wildtype (WT) or TNF‐α–deficient (KO) mice. The left incisor was extracted 4 days after LPS injection. The measuring area was established as the tooth socket under the mesial root of the first molar. A significant increase in serum TNF‐α levels after LPS injection was observed in WT mice. The BMD of the tooth socket was significantly decreased by LPS injection 21 days after extraction in WT but not in KO mice. Histomorphometric analysis showed a significant decrease in the mineral apposition rate after LPS injection, which appeared at an early stage in WT but not in KO mice. Injection of a peptide that blocked the TNF‐α signaling pathway by preventing transmission of the NF‐κB signal recovered the inhibition of osteogenesis observed after LPS injection. In conclusion, TNF‐α might play a major role in LPS‐induced inhibition of osteogenesis under inflammatory conditions.     

Expression of human β‐defensin‐2 in the prostate

What’s known on the subject? and What does the study add? We found the expression of human β‐defensin‐2 (HBD‐2) in the prostate for the first time and LPS, a gram negative bacterial component, upregulated HBD‐2 in prostate epithelial cells. We are looking for other antimicrobial peptides expressed in the prostate besides human β‐defensin‐2. Also, we are studying the relationship between antimicrobial peptides and the development or progression of prostate diseases.

OBJECTIVE

To investigate the expression and regulation of human β‐defensin‐2 (HBD‐2) in the prostate.

PATIENTS AND METHODS

Normal human prostate epithelial cell line (RWPE‐1), human prostate cancer cell lines (DU‐145, PC‐3), and paraffin‐embedded prostate tissue from patients with benign prostatic hyperplasia (BPH) were analysed by RT‐PCR and immunohistochemical staining. HBD‐2 expression was also analysed by RT‐PCR and ELISA in RWPE‐1 cells treated with lipopolysaccharide (LPS). Nuclear factor‐κB (NF‐κB) activation was assessed by IκBα immunoblotting and electrophoretic mobility shift assay (EMSA).

RESULTS

BPH tissue and all of the tested prostate cell lines other than PC‐3 constitutively express HBD‐2 mRNA. HBD‐2 protein was strongly detected in prostate gland tissue surrounded by inflammatory cells including macrophages. Exposure to LPS induced HBD‐2 upregulation and NF‐κB activation, as assessed by IκBα phosphorylation and degradation in RWPE‐1 cells. Bay11‐7082, an NF‐κB inhibitor prevented LPS‐induced HBD‐2 production in RWPE‐1 cells.

CONCLUSIONS

Prostate epithelial cells may constitutively express HBD‐2, and its expression was upregulated by LPS. Our data indicate that HBD‐2 may be an important immunomodulatory factor in prostate function. Expression of HBD‐2 in normal prostates and the potential role of HBD‐2 in prostatitis and BPH should be addressed in the future.     

Processing of the NF‐κB2 precursor p100 to p52 is critical for RANKL‐induced osteoclast differentiation

Gene targeting of the p50 and p52 subunits of NF‐κB has shown that NF‐κB plays a critical role in osteoclast differentiation. However, the molecular mechanism by which NF‐κB regulates osteoclast differentiation is still unclear. To address this issue, we analyzed alymphoplasia (aly/aly) mice in which the processing of p100 to p52 does not occur owing to an inactive form of NF‐κB‐inducing kinase (NIK). Aly/aly mice showed a mild osteopetrosis with significantly reduced osteoclast numbers. RANKL‐induced osteoclastogenesis from bone marrow cells of aly/aly mice also was suppressed. RANKL still induced the degradation of IκBα and activated classical NF‐κB, whereas processing of p100 to p52 was abolished by the aly/aly mutation. Moreover, RANKL‐induced expression of NFATc1 was impaired in aly/aly bone marrow. Overexpression of constitutively active IKKα or p52 restored osteoclastogenesis in aly/aly cells. Finally, transfection of either wild‐type p100, p100ΔGRR that cannot be processed to p52, or p52 into NF‐κB2‐deficient cells followed by RANKL treatment revealed a strong correlation between the number of osteoclasts induced by RANKL and the ratio of p52 to p100 expression. Our data provide a new finding for a previously unappreciated role for NF‐κB in osteoclast differentiation. © 2010 American Society for Bone and Mineral Research     

Combined effects of TNF‐α, IL‐1β, and HIF‐1α on MMP‐2 production in ACL fibroblasts under mechanical stretch: An in vitro study

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra‐articular joint, are very complex after injury. Injury to intra‐articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro‐inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix‐metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP‐2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor‐1α (HIF‐1α) on induction of MMP‐2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF‐α and IL‐1β elevate the activity of MMP‐2 in a dose‐ and time‐dependent manner. In addition, mechanical stretch further enhances the MMP‐2 protein levels with TNF‐α, IL‐1β, and their mixture. CoCl₂‐induced HIF‐1α (100 and 500 µM) also increases the levels and activity of MMP‐2. Mechanical stretch has a strong additional effect on MMP‐2 production with HIF‐1α. Our results conclude that mechanical injury, HIF‐1α and inflammatory factors collectively induce increased MMP‐2 production in ACL fibroblasts, which was inhibited by NF‐κB pathway inhibitor (Bay‐11‐7082). © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1008–1014, 2011     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three‐dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF‐β pathways. We compared the effects of DBP, TGF‐β1, or BMP‐2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT‐PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP‐2, and likewise DBP effects on Wnt genes and β‐catenin only partially required the TGF‐β pathway, as shown by selective inhibition of TGF‐β/activin receptor‐like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP‐2 and by TGF‐β1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF‐β pathways. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 554–560, 2013     

Biphasic effects of interleukin‐1β on osteoblast differentiation in vitro

A rat calvarial cell model of osteoblast differentiation using the formation of bone nodules in vitro as an endpoint was used to assess the effects of IL‐1β on osteoblast differentiation. Short‐term treatment (2 days) with IL‐1β early in culture resulted in increased nodule number and size as well as calcium content in contrast to long‐term treatment (6 days) in cultures assessed at 10–12 days. This increase in bone formation was blocked by IL‐1 receptor antagonists. Short‐term treatment increased COX‐2, prostaglandin (PGE₂), and iNOS production. Exogenous PGE₂ with IL‐1β enhanced this effect. COX‐2 inhibitors, indomethacin and N‐39, blocked 50% of nodule formation. NO donor did not modify effects of IL‐1β, but iNOS inhibitor (1400W) partially blocked the effects. However, PGE₂ and NO donors could not rescue the decreased nodule number resulting from long‐term IL‐1β treatment. The results of this study suggest a biphasic effect of IL‐1β on bone nodule formation activated by IL‐1β binding with IL‐1 receptors, and the anabolic effect of early short‐term treatment with IL‐1β is likely mediated by PGE without ruling out nitric oxide. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:958–964, 2010